Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe–S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.


Results
Differentiated HUDEP-2 cells stably expressing SF3B1 K700E exhibited RS formation. To examine the direct link between the expression of SF3B1 K700E and RS formation, we established HUDEP-2 cells stably expressing SF3B1 K700E , which were subsequently induced to undergo erythroid differentiation by co-culturation with OP-9 cells (see Supplementary Fig. S1a-b online). Control vector-transduced HUDEP-2 cells and HUDEP-2 cells stably expressing SF3B1 WT were used as controls. Expression of codon optimized SF3B1 WT or SF3B1 K700E were confirmed by RT-PCR and Sanger sequencing (see Supplementary Fig. S1c and S3 online).
Quantitative RT-PCR revealed specifical expression of codon optimized SF3B1 in HUDEP-2 cells stably expressing SF3B1 WT or SF3B1 K700E and global expression of internal SF3B1 in each cell line, although the expression of level of internal SF3B1 was significantly higher in HUDEP-2 cells stably expressing SF3B1 K700E when compared to controls (see Supplementary Fig. S4 online). The relatively low expression level of codon optimized SF3B1 K700E compared to that of codon optimized SF3B1 WT might imply the survival inferiority of HUDEP-2 cells expressing SF3B1 K700E at a high level. The higher level of internal SF3B1 in HUDEP-2 stably expressing SF3B1 K700E than in controls could be explained as compensatory mechanism for inhibiting mis-splicing caused by abnormal SF3B1 in HUDEP-2 cells stably expressing SF3B1 K700E . May-Grünwald-Giemsa staining confirmed erythroid differentiation into polychromatic and orthochromatic erythroblasts (Fig. 1a). The proportion of RS was higher in HUDEP-2 cells stably expressing SF3B1 K700E than in controls (Fig. 1a, b). Electron microscopic observation of HUDEP-2 cells stably expressing SF3B1 K700E revealed mitochondria containing electron-dense deposits, indicating abnormal iron accumulation (Fig. 1c).
Gene expression analysis for HUDEP-2 cells stably expressing SF3B1 K700E . To detect candidate genes contributing to RS formation in HUDEP-2 cells stably expressing SF3B1 K700E , we performed RNA-seq analysis based on HUDEP-2 cells stably expressing SF3B1 K700E and controls. Comprehensive gene expression analysis with RNA-seq did not show apparent changes in HUDEP-2 cells stably expressing SF3B1 K700E in the expression levels of CSA-causative genes, except ALAS2 (see Supplementary Table S1 online). Western blotting revealed downregulation of ALAS2 and ABCB7 in HUDEP-2 cells stably expressing SF3B1 K700E (Fig. 2a). Quantitative RT-PCR demonstrated significantly decreased expression levels of ABCB7, ALAS2 and GLRX5 (Fig. 2b). These results suggested the contribution of some dysregulated CSA-causative genes to RS formation in MDS-RS.
Moreover, comprehensive gene expression analysis with RNA-seq revealed downregulation of GATA-1 target genes, including ALAS2, solute carrier family 4 member 1 (SLC4A1) 27 , ankyrin-1 (ANK1) 28 , and aminolevulinate dehydrogenase (ALAD) 29 in HUDEP-2 cells stably expressing SF3B1 K700E (see Supplementary Table S3 online). Decreased expression levels of mitogen-activated protein kinase 7 (MAP3K7) and GATA-1 in HUDEP-2 cells stably expressing SF3B1 K700E were described by quantitative RT-PCR and western blotting (Fig. 2c, d). In contrast, quantitative RT-PCR for differentiated HUDEP-2 cells did not show dysregulation of ALAS2, MAP3K7, ABCB7, GATA-1 and GLRX5 by SF3B1 K700E expression (see Supplementary Fig. S5   www.nature.com/scientificreports/ imide (CHX) treatment (Fig. 3). These results indicated the existence of SF3B1 K700E -induced A3SS events as previously reported 19,33 , leading to the production of splice variants targeted by nonsense-mediated decay (NMD). Moreover, we performed comprehensive alternative splicing (AS) analysis using Mixture of Isoforms (MISO) 34 . MISO analysis did not detect significant differences in the number of significant AS events between HUDEP-2 cells stably expressing SF3B1 WT and SF3B1 K700E when compared with control vector-transduced HUDEP-2 cells (see Supplementary Fig. S12a, Tables S3-4 online). Significant AS events in HUDEP-2 cells stably expressing SF3B1 K700E were not detected in ALAS2 and GLRX5, suggesting that these genes would not be downregulated through aberrant splicing induced by SF3B1 K700E (see Supplementary Tables S4 online). Although the number of A3SS events detected in HUDEP-2 cells stably expressing SF3B1 WT or SF3B1 K700E was equivalent, most of the events did not overlap. HUDEP-2 cells stably expressing SF3B1 K700E exhibited the significant A3SS event in ribonuclease/angiogenin inhibitor 1 (RNH1) (see Supplementary Fig. S12b online) 33 . Although aberrantly   www.nature.com/scientificreports/ spliced isoforms were detected even in control vector-transduced HUDEP-2 cells by RT-PCR, the sashimi plot described by MISO revealed a significant increase in A3SS usage between exons 2 and 3 of RNH1 in HUDEP-2 cells stably expressing SF3B1 K700E (see Supplementary Fig. S13a-b online). Sanger sequencing of RT-PCR products revealed that A3SS caused the addition of 68 bases within the 5′ untranslated region (UTR) of RNH1 (see Supplementary Fig. S13c online). However, we could not detect any difference in the expression level of RNH1 either at mRNA or at protein level between HUDEP-2 cells stably expressing SF3B1 K700E and controls (see Supplementary Fig. S13d-e online). Thus, we demonstrated that ABCB7 could be mainly downregulated via increased A3SS usage between exons 8 and 9 induced by SF3B1 K700E . Moreover, downregulation of ALAS2 at the mRNA level was suggested to be associated with decreased GATA-1 function, probably caused by downregulation of MAP3K7 because of increased A3SS usage between exons 4 and 5.
Confirmation of mis-splicing-mediated downregulation of ABCB7 and MAP3K7 in K562 cells. Although downregulation of MAP3K7 and ABCB7 possibly caused by mis-splicing was detected in HUDEP-2 cells stably expressing SF3B1 K700E , the incidence of A3SS events in MAP3K7 and ABCB7 was so low that the detection of A3SS events required NMD inhibition with CHX. To reinforce the evidence for missplicing-mediated downregulation of ABCB7 and MAP3K7 induced by SF3B1 K700E , K562 cells overexpressing SF3B1 K700E or SF3B1 WT were generated by electroporation (see Supplementary Fig. S16 online), as confirmed using western blotting (Fig. 4a). Repeated electroporation resulted in stronger expression of SF3B1 in K562 cells expressing SF3B1 WT or SF3B1 K700E than in HUDEP-2 cells stably expressing SF3B1 WT or SF3B1 K700E . Similar to the experiment with HUDEP-2 cells (Fig. 2), quantitative RT-PCR and western blotting confirmed significantly decreased expression levels of ABCB7 and MAP3K7 both at mRNA and protein levels in K562 cells expressing SF3B1 K700E compared to those in control vector-transduced K562 cells or K562 cells expressing SF3B1 WT (Fig. 4a, b). The number of significant AS events detected by MISO analysis was higher in K562 cells expressing SF3B1 K700E than in those expressing SF3B1 WT when compared with control vector-transduced K562 cells (Fig. 4c). Lists of significant AS events can be found in Supplementary Table S5-6. Read-coverage visualization with IGV detected A3SS events between exons 4 and 5 of MAP3K7 and between exons 8 and 9 of ABCB7 in K562 cells expressing SF3B1 K700E even without CHX treatment (Fig. 4d, e).

ABCB7-knockdown in HUDEP-2 cells lead to RS formation.
Although ABCB7 is known to be one of the genes responsible for CSA 1 , the direct contribution of ABCB7 defects to RS formation has not been demonstrated. Thus, we conducted shRNA-mediated ABCB7-knockdown in HUDEP-2 cells, which were subsequently induced to undergo erythroid differentiation (see Supplementary Fig. S22 online). ABCB7-knockdown was confirmed both at mRNA and protein levels in undifferentiated HUDEP-2 cells, especially using shRNA clone 5 ( Fig. 5a, b). Decreased expression level of ABCB7 in differentiated ABCB7-knockdown HUDEP-2 was confirmed by quantitative RT-PCR, unless western blotting could not be performed (Fig. 5a). Although there was no significant change in ALAS2 mRNA expression levels (Fig. 5c), ALAS2 protein levels were noticeably decreased by ABCB7-knockdown, indicating decreased ALAS2 translation (Fig. 5b). The progression of erythroid differentiation was also morphologically confirmed by May-Grünwald-Giemsa staining (Fig. 5d, upper panel). Prussian blue staining revealed an increased frequency of RS formation in ABCB7-knockdown HUDEP-2 cells compared with that in control shRNA-transduced HUDEP-2 cells (Fig. 5d, lower panel). Expression profiling analysis revealed that 39 and 20 genes were commonly upregulated and downregulated by more than 1.5-fold, respectively, in ABCB7-knockdown HUDEP-2 cells compared with control cells (see Supplementary Table S7 online). Gene ontology (GO) enrichment analysis by Metascape revealed significant enrichment of genes involved in iron metabolism and apoptosis among the commonly upregulated and downregulated genes, respectively (Fig. 6).
Collectively, we demonstrated a direct link between ABCB7 defects and RS formation, which might be associated with decreased ALAS2 translation.  Fig. S26 online), indicating a greater contribution of SF3B1 mutation status, rather than RS existence, to the regulation of ABCB7 and MAP3K7 expression. RNA-seq read-coverage of ABCB7 and MAP3K7 visualized by IGV revealed previously reported AS events in some SF3B1 MUT -MDS patients (Fig. 7b, c), although the incidence of A3SS between exons 8 and 9 of ABCB7 seemed very low. AS events in ABCB7 and MAP3K7 accompanied with lower expression levels of ABCB7 and MAP3K7 in SF3B1 MUT -MDS patients indicate downregulation of ABCB7 and MAP3K7 due to degradation of mis-spliced transcripts by NMD. Additionally, the A3SS event between exons 2 and 3 of RNH1 detected in HUDEP-2 cells expressing SF3B1 K700E was confirmed in both SF3B1 WT -and SF3B1 MUT -MDS patients, whereas the PSI of this event was relatively high in SF3B1 MUT -MDS patients (See Supplementary Fig. S27 online).

Analysis of gene expression and AS
Lastly, we analyzed RNA-seq dataset GSE114922 obtained from GEO RNA-seq Experiments Interactive Navigator (GREIN) 35     www.nature.com/scientificreports/ Similar to our findings (Fig. 7), the expression levels of ABCB7 and MAP3K7 were significantly lower in SF3B-1 MUT -MDS patients than in SF3B1 WT -MDS patients, which was also confirmed for the cohort excluding CMML (Chronic myelomonocytic leukemia) and RAEB (Refractory anemia with excess blasts) (see Supplementary  Fig. S28 online). Furthermore, we aimed to find the unique role of SF3B1 K700E as compared with SF3B1 non-K700E . The expression levels of ABCB7 and MAP3K7 were compared among SF3B1 WT -, SF3B1 K700E   www.nature.com/scientificreports/ were downregulated) and 27 (2 genes were upregulated and 25 genes were downregulated), respectively (see Supplementary Fig. S31 online). Gene ontology (GO) enrichment analysis by Metascape 38 revealed significant enrichment of the genes involved in oxygen transport and erythroid differentiation among the upregulated genes and those involved in the productivities or responsivities of cytokines among the downregulated genes in SF3B1 K700E -MDS patients (see Supplementary Fig. S32 online). These data imply that SF3B1 K700E may exert more comprehensive impact on gene expression than SF3B1 non-K700E .

Discussion
In this study, we successfully established HUDEP-2 cells expressing SF3B1 K700E as an MDS-RS model (Fig. 1). This model exhibited downregulation of ABCB7, ALAS2, and GLRX5 (Fig. 2), all of which are known CSA-causative genes, indicating a cross-link between acquired and congenital SA. www.nature.com/scientificreports/ ABCB7, located at Xp13.3, encodes a mitochondrial transporter of the Fe-S cluster and is responsible for XLSA with ataxia (XLSA/A) 1 . The mis-splicing-associated downregulation of ABCB7 detected in our MDS-RS model (Figs. 2a, b, 3a) is closely associated with MDS-RS 5,18 . As Abcb7-knockout mice do not survive 39 , RS formation by ABCB7 defects in erythroid cells has never been reproduced. Here, we firstly established ABCB7-deficient RS model by ABCB7-knockdown in HUDEP-2 cells (Fig. 5). This model showed decreased expression levels of ALAS2 at the protein level, but not at the mRNA level (Fig. 5b, c), indicating impaired ALAS2 translation by ABCB7-knockdown. ALAS2 translation is inhibited when iron regulatory protein 1 (IRP1) binds iron-responsive element (IRE) located in the 5′ UTR of ALAS2 mRNA, but is promoted when IRP1 combined with Fe-S cluster is converted to aconitase lacking the ability to bind IRE 40 ; hence, we speculated that a decrease in cytosolic Fe-S cluster owing to downregulation of ABCB7 could contribute to RS formation by inhibiting ALAS2 translation via IRP1 activation. Further analyses are required to assess changes in aconitase activity and IRP/IRE interactions.
The molecular link between ALAS2 defects and RS formation has already been previously demonstrated 24,25 . Comprehensive AS analysis for HUDEP-2 cells stably expressing SF3B1 K700E did not detect any AS events in ALAS2 (see Supplementary Table S4 online), indicating unlikeliness of mis-splicing-mediated downregulation of ALAS2. However, we observed downregulation of GATA-1 target genes, such as ALAS2 8 , SLC4A1 26 , ANK1 27 , and ALAD 29 , and confirmed decreased GATA-1 protein levels (Fig. 2c). Thus, we focused on MAP3K7 as the cause of GATA-1 dysregulation in our MDS-RS model, because MAP3K7, which is activated by TGF-β, is known to phosphorylate p38MAPK, regulating GATA-1 function both by phosphorylating GATA-1 and by promoting ubiquitination and proteasomal degradation of GATA-1 via MAPKAKP2/HSP27 30,31 . Our MDS-RS model exhibited mis-splicing-associated downregulation of MAP3K7 (Figs. 2c, d, 3b), which was confirmed in K562 cells overexpressing SF3B1 K700E (Fig. 4a, b and e) and SF3B1 MUT -MDS patients (Fig. 7a and c). Downregulation of MAP3K7 mediated by mis-splicing has been reported to decrease the expression level of GATA-1 in SF3B1 K700E -mutated K562 cells 33 . Additionally, our MDS-RS model showed increased PSI of the A3SS event within 5′ UTR of RNH1 (see Supplementary Fig. S13 online), which was also confirmed in SF3B1 MUT -MDS patients (see Supplementary Fig. S27 online). RNH1 could be broadly detected in various human tissues including enucleated erythroid cells lacking RNase 41 . RNH1 has been reported not only as the indispensable factor for survival and development of mice due to its protective role of global RNA from RNase but also as the translational regulator for the specific genes including GATA-1 42,43 . We hypothesized that impaired RNH1 translation associated with increased A3SS usage within 5′ UTR induced by mutant SF3B1 might contribute to the downregulation of ALAS2 at the transcription level by enhancing impaired GATA-1 translation in SF3B1 MUT -MDS. Although we failed to demonstrate decreased RNH1 translation followed by impaired GATA-1 translation in HUDEP-2 stably expressing SF3B1 K700E , this could be confirmed by a novel approach like polysome profiling or western blotting for each fraction of the cells. Taken together, we speculate that ALAS2 downregulation mediated by GATA-1 dysfunction may play a role in RS formation induced by SF3B1 K700E expression.
Our MDS-RS model exhibited GLRX5 downregulation (Fig. 2b). Comprehensive AS analysis did not indicate mis-splicing-mediated downregulation of GLRX5 (see Supplementary Table S4 online). Although the mechanism by which downregulation of GLRX5 was induced by SF3B1 K700E is unknown, it might be mediated by the downregulation of ABCB7 because ABCB7-knockdown downregulates GLRX5 44 . The role of GLRX5 as a supplier of Fe-S cluster to IRP1 45 suggests that downregulation of GLRX5 could also inhibit ALAS2 translation via IRP1 activation, such as downregulation of ABCB7.
The inner mitochondrial membrane protein TMEM14C which functions in the final steps of heme synthesis as the transporter of protoporphyrinogen IX into mitochondrial matrix is highly expressed in the erythroid cells at the terminal differentiation steps 46 . TMEM14C expression is not under the control of IRP-IRE system 46 , while transcriptionally regulated by GATA-1 8 . According to iPSC-derived SF3B1 G742D -MDS-RS model, coordinated mis-splicing TMEM14C and ABCB7 contributed to RS formation 21 . In our models, increased frequency of the A3SS event was observed in K562 overexpressing SF3B1 K700E and some SF3B1 MUT -MDS patients, but not in HUDEP-2 stably expressing SF3B1 K700E (see Supplementary Fig. S33-34 online). We speculated that lower expression level of SF3B1 K700E compared to endogenous SF3B1 (see Supplementary Fig. S4 online) might make it difficult to detect obvious TMEM14C mis-splicing. Further analysis based on more physiological model, such as CRISPR/Cas9-mediated SF3B1 mutation in HUDEP-2 cells, would be preferred to demonstrate the detailed mechanism of RS formation by SF3B1 mutation.
In conclusion, our findings have delineated a complex mechanism for RS formation, including dysregulation of ABCB7, ALAS2, GATA-1, and MAP3K7 expression (Fig. 8). Additionally, according to the observations on ABCB7-knockdown HUDEP-2 cells, downregulation of ABCB7 could impair ALAS2 translation presumably through via IRP1 activation. These results imply a complicated mechanism for RS formation in SF3B1 MUT -MDS. Further characterization of the established MDS-RS model will aid in clarifying its molecular etiology and establishing novel therapeutic strategies.

Methods
Ethical statement. Informed consent was obtained from all patients. The protocol of this study was approved by the Institutional Review Board of Tohoku University Graduate School of Medicine and was based on the ethical principles for medical research involving human subjects of the Helsinki Declaration.

RS formation
GLRX5 mRNA mitochondrial iron accumulation Figure 8. Proposed mechanism for RS formation in SF3B1 MUT -MDS. A3SS usage in ABCB7, MAP3K7, and RNH1 is promoted by the spliceosome containing the mutant SF3B1. Targeting of increased mis-spliced ABCB7 mRNA by NMD contributes to the downregulation of ABCB7, resulting in reduced translation of ALAS2 through an enhanced IRP1-IRE system induced by reduced cytosolic Fe-S cluster. Increased mis-spliced MAP3K7 mRNA, also targeted by NMD, contributes to the downregulation of MAP3K7, causing deterioration of GATA-1 function through the reduction in phosphorylated p38MAPK, as previously described 31 . Missplicing in the 5′ UTR of RNH1 might impair translation of RNH1, leading to downregulation of RNH1, which was reported to downregulate GATA-1 by inhibition of translation 42 . Thus, transcription of ALAS2 is reduced owing to decreased GATA-1 function induced by downregulation of MAP3K7, and perhaps RNH1.
In conclusion, downregulation of ALAS2 at both the transcriptional and translational levels because of mutant SF3B1-induced mis-splicing in ABCB7, MAP3K7, and possibly RNH1 could be considered the underlying mechanism of RS formation in SF3B1 MUT -MDS. Moreover, downregulation of ABCB7 and GLRX5 also promoted RS formation by accelerating mitochondrial iron accumulation through decreased Fe-S cluster export from the mitochondria and decreased production of Fe-S cluster, resulting in decreased use of mitochondrial free iron. p-p38MAPK indicates phosphorylated p38MAPK.  49 , encoded in pcDNA (Invitrogen, Carlsbad, CA). Each coding sequence was cloned into the retroviral vector pBABE-puro (Addgene Plasmid #1764) 50 .
For transient overexpression of SF3B1 WT or SF3B1 K700E , each pcDNA-based expression vector (10 μg) was transfected into K562 cells using the Amaxa Cell Line Nucleofector II (Lonza, Cologne, Germany) with the program T-016 27,47 . For retroviral overexpression of SF3B1 WT or SF3B1 K700E , the pBABE-puro-based expression vector and VSV-G (Addgene plasmid #12259) were co-transfected into Plat-GP packaging cell lines (Cell Biolabs, San Diego, CA) using FuGene HD (Promega, Madison, WI). Seventy-two hours after transfection, viral supernatant was used for infection. After spin infection of HUDEP-2 cells at 1300 × g for 2 h, 1 μg/mL puromycin (Sigma-Aldrich) was added to the medium to select the transduced cells.
Primers used for amplification or detection of codon-optimized SF3B1 WT or SF3B1 K700E expression are shown online in Supplementary Table S9.
Detection of SF3B1 mutation. Genomic DNA was extracted from whole BM lysates of MDS patients or cell lysates of SF3B1 mutant cell lines using a DNeasy Blood & Tissue kit (Qiagen N.V., Hulsterweg, Netherlands). Mutations within SF3B1 exons 14 to 16, where most SF3B1 mutations exist, were screened using highresolution melting analysis as previously reported 51 , followed by confirmation of the mutations with Sanger sequences when screening tests were positive. The primer sequences used for Sanger sequencing are listed online in Supplementary Table S9. shRNA-mediated ABCB7-knockdown in HUDEP-2 cells. Lentiviral-based knockdown of the human ABCB7 gene was conducted with pGIPZ lentiviral shRNAmir (Clone ID: V3LHS_406787) (Open Biosystems, Huntsville, USA), as described previously 47 . The lentiviral vectors VSV-G and psPAX2 (Addgene plasmid #12260) were co-transfected into HEK293T cells; 72 h after transfection, the viral supernatant was used for infection, as in the retroviral overexpression protocol.
Quantitative RT-PCR. Quantitative RT-PCR were conducted as described previously 27 . Primers used for quantitative RT-PCR are listed online in Supplementary Table S9.
Expression profiling analysis. For RNA-seq analysis, total RNA was extracted from whole BM cells of MDS patients (see Supplementary Table S8 online) or cell pellets when analyzing cell lines using TRIzol reagent. For library preparation, the SMARTer Ultra Low RNA Kit (Illumina, San Diego, CA) and Illumina TruSeq stranded mRNA Library kit were used for clinical samples and the NEBNext Ultra II RNA Library Prep Kit for Illumina was used for cell lines. Libraries were sequenced on an Illumina NovaSeq6000 (Otogenetics, Norcross, GA, USA). Sequence data were mapped to the human reference genome, hg19/GRCh37, using HISAT2 (version 2.2.1) (http:// daehw ankim lab. github. io/ hisat2/) 52 . Normalized expression level of each gene was calculated as transcripts per million using StringTie (version 2.1.7) (https:// ccb. jhu. edu/ softw are/ strin gtie/) 53 .
Microarray analysis was conducted using a Human Oligo chip 25 k (Toray, Tokyo, Japan), and subsequent GO enrichment analyses were performed using Metascape 38 . For global normalization, the background value was subtracted and subsequently adjusted to an average signal value of 25, and genes with > 100 were analyzed.
DEGs analysis for GSE114922 dataset was performed with web tool, iDEP.951 (http:// bioin forma tics. sdsta te. edu/ idep/) 37 based on normalized count data obtained from GREIN. We selected DESeq2 method for DEGs identification setting false discovery rate cut off at 0.05 and a minimum fold change at 1.5.
Alternative splicing analysis. The AS events of each sample were analyzed using MISO software (version 0.5.4) (https:// miso. readt hedocs. io/ en/ fastm iso/) with exon-centric analysis separately for A3SS, alternative 5′ splice site (A5SS), mutually exclusive exons (MXE), retained introns (RI) and skipping exons (SE) 34 . Human genome (hg19) alternative events v2.0 (https:// miso. readt hedocs. io/ en/ fastm iso/ annot ation. html), was used for MISO annotation. Among AS events extracted by the "compare miso" command, we considered the AS events passing all filtering criteria as significant, which in our study is as follows: (a) at least one inclusion read and one exclusion read, such that (b) the sum of inclusion and exclusion reads is at least 10, (c) the Δ PSI (percent spliced in) is ≥ 0.20, and (d) the Bayes factor is at least 10. AS events were expressed with visualized read-coverage using IGV 32 or the Sashimi plot command in IGV or MISO.
Cycloheximide treatment. As splice variant isoforms containing premature stop codons (PTCs) are targeted by NMD, which decreases the number of splice variant isoforms 54 , the less expressed splice variant isoforms are sometimes difficult to detect. Cycloheximide (Nacalai Tesque, Inc., Japan), an NMD inhibitor, was added to HUDEP-2 cells stably expressing SF3B1 WT or SF3B1 K700E at a final concentration of 100 ug/mL 19 .